Instructors and Employees

Alumni

Martin Røssel Larsen

The group of Martin R. Larsen is focusing on the application of biological mass spectrometry in proteomics, especially the characterization of post-translational modifications in proteins and their influence on biological systems. The group is working with a large variety of biological systems and human diseases through collaborations with both academics on Universities and hospitals, and industry.

The primary focus in the group is to develop new and efficient quantitative mass spectrometric and affinity chromatographic strategies for the characterization of phosphorylated and glycosylated proteins from highly complex mixtures and apply these strategies to the study of various biological systems. The current larger projects in the group are:

- Development of large scale quantitative strategies for the identification and characterization of phosphorylated and sialylated proteins from low amount of cell material (e.g., primary cells).

- Characterization of the phosphorylation-dependent calcium signaling in nerve terminals after various stimuli, i.e., depolarization.

- Characterization of phosphorylation dependent signaling events in pancreatic beta-cells exposed to various pro-inflammatory cytokines such as Interleukine-1β.

- Characterization of altered glycosylation (i.e., sialylation) on surface proteins of cancer cells and their influence on metastasis.

- Characterization of microparticles and exosomes from apoptotic cells and body-fluids

- Membrane proteomics and the characterization of phosphorylated and sialylated proteins in membranes.

- Kinomics - the study of kinases and their presence and activity in the cell using kinase inhibitor affinity purification and mass spectrometry. Characterization of kinases in nerve-terminals.

- Biomarker discovery with the focus on glycosylated proteins in various body-fluids such as plasma, serum and cerebrospinal fluid (type 2 diabetes, Alzheimers Disease and other CNS diseases)

Beside the above mentioned projects we have a large number of small projects mainly through external collaborations.

For more information on the different areas and projects please see extended CV of Martin R. Larsen

1: Methodological development for modification specific proteomics on biological systems.

My research team is at the cutting edge of method development for characterizing PTMs such as phosphorylation and glycosylation in immortalized cells in culture. Having developed several methods for assessing modified peptides from complex mixtures lately we are very confident that we only observe the tip of the iceberg and do not detect the transient or low abundant biologically important modifications. In addition, recent results obtained in my group suggest that the current methods for PTMs characterization are not applicable for true in vivo studies using primary cells originating directly from animals or humans. For example investigating the phosphoproteome of a cell after external stimulation are mostly performed only using cell cultures that have been serum-starved to reduce the basal phosphorylation caused by growth factors in the medium. Serum-starvation is frequently not applicable to primary cells and tissues which undergo apoptosis under such culture conditions. Other factors also influence the analysis of primary cells and tissues, including the presence of extracellular matrix, mixed cell populations and the significantly reduced available sample for microanalysis. We speculate that not only phosphorylation but all kind of PTMs will be much more challenging to characterize from true in vivo biological systems and that current methods for analyzing PTMs are not appropriate for true in vivo studies. We have therefore initiated a focused research program aiming at developing new and more sensitive mass spectrometric methods for the assessment of PTMs from primary cells, tissues and body-fluids, biological material that cannot be manipulated prior to analysis. We will combine the newly developed affinity enrichment methods with extensive multidimensional separation methods in order to achieve the most sensitive comprehensive technique for phosphoproteimics and glycomics.

Another problem in current PTM analysis is the quantitative assessment of different PTMs in proteins. After identifying for example phosphorylation sites, the next obvious step is to quantify the changes in phosphorylation in different situations. We will continue optimizing methods for more complex samples and develop new methods for accurate quantification of PTMs from very small amount of material using stabile isotope labelling in combination with nano-purification techniques and accurate mass spectrometry.

2: Characterization of the molecular mechanism of Synaptic Vesicle Endocytosis.

There are many billions of neurons in the human brain, each with the ability to influence many other cells. An average neuron will make approximately 10,000 contacts, or synapses, with other neurons while receiving a similar input itself. Therefore, sophisticated mechanisms are essential to enable efficient communication within this highly complex system. These mechanisms are functionally localized at synapses which are the contact points between neurons. The presynaptic side of the synapse (the presynaptic nerve terminal) is the focus of this study. Although many kinds of synapses exist within the brain, they can be divided into two distinct populations: electrical synapses and chemical synapses. In the brain, the number of electrical synapses is significantly lower than the number of chemical synapses. Electrical synapses permit direct, passive flow of electrical current from one neuron to another and therefore allow much faster communication than chemical synapses. In contrast, chemical synapses use small signalling molecules, or neurotransmitters (e.g. acetylcholine, dopamine or glutamate), to enable cell-to-cell communication. Neurotransmitters are stored in small organelles termed synaptic vesicles (SVs), which are normally at rest bound to the cytoplasmic cytoskeleton. Upon depolarization, neurotransmitters are released by the presynaptic terminal into the space between the pre- and postsynaptic cells, the synaptic cleft, and bind and activate specific receptors at the post-synaptic membrane to initiate signalling events. After release of neurotransmitters the SV is retrieved back into the nerve terminal. This overall process is called synaptic transmission.

In the chemical synapse, the release of neurotransmitters is triggered by the influx of calcium through ion-channels in the presynaptic membrane. An increase of Ca2+ in the presynaptic nerve terminal leads to a multitude of molecular events resulting in release of neurotransmitters to the synaptic cleft. SVs are firstly released from the cytoskeleton and transported to the plasma membrane at the synapse where they fuse with the presynaptic plasma membrane to release their content into the synaptic cleft – this is called exocytosis. After release of the neurotransmitter molecules, the used SVs are retrieved, also via a Ca2+-regulated process, back into the pre-synaptic cell for another round of neurotransmitter filling and exocytosis – this is called endocytosis. Both exo- and endocytosis are controlled by phosphorylation and dephosphorylation of synaptic proteins by various kinases and phosphatases that become activated as a result of Ca2+ influx.

Previously we have in close collaboration with Prof Phillip J. Robinson, Children’s Medical Research Institute (CMRI), Sydney, Australia found more than 50 phosphorylation sites on 4 different proteins that participate in the SV endocytosis. In future projects we will (1) complete a full map of phosphorylated proteins in the synaptosomes and quantify their changes during depolarization, (2) elucidate the biological role of these phosphorylation sites in protein interaction and complex assembly, (3) quantify the changes of phosphorylation of synaptosomal proteins in neurological diseases e.g., epilepsy.

3: Unrevealing the pathogenesis of Type 1 Diabetes Mellitus.

Accumulating evidence supports a role for cytokines, especially interleukin 1b (IL-1b), released during this inflammation, in the pathogenesis of T1DM. IL-1b, either alone, or in combination with other cytokines (tumour necrosis factor-a and interferon g) induces the production of toxic nitric oxide (NO) free radicals by the beta-cell itself, which leads to beta-cell self-destruction. We have initiated a research project elucidating the phosphorylation events involved in interleukin 1b signal transduction. We have discovered from low amount of material a large number of phosphorylated peptides and new phosphorylation sites that have not been characterized and quantified before. This study will be extended to elucidate the phosphorylation events in the signal transduction pathways of the three pro-inflammatory cytokines; IL-1b, TNFa and INFg and the crosstalk between those pathways.

Knowledge on the exact molecular mechanisms used by each cytokine in signalling cascades is still very limited. The phosphorylation events of the proteins in the downstream signalling pathways (STAT1, IKK-NFκB and MAPK) are extensively characterized primarily because they are shared by many other signalling pathways (e.g., EGF and PDGF). However, information on the regulatory phosphorylation events of the proximal membrane complexes are relatively limited. Knowledge of the precise regulation of these complexes by phosphorylation is essential to the future development of new pharmaceuticals designed to stop the development of T1DM by blocking the phosphorylation dependent activation (e.g., by inhibiting an involved kinase). Drugs inhibiting proteins later in the pathway will consequently effect many other functions in the cell and thus cause many unwanted side effects.

4: Investigation of the influence of altered protein glycosylation on cancer development and metastasis.

Protein glycosylation is among the most common PTMs known in nature. Glycosylation is difficult to analyze by any biochemical methods due to the chemically very similar monosaccharide building blocks and pronounced glycosylation site heterogeneity and micro-heterogeneity of the carbohydrate chains with respect to branching patterns and monosaccharide composition. The biological role of protein glycosylation varies from conformational stability, protection against degradation, to molecular and cellular recognition in for example development, growth and cellular communication.

It is well known that glycosylation patterns in chronic disease can be highly aberrant as a consequence of changes in the expression or activity of glycosyltransferases or other factors affecting the glycan biosynthesis. Many extracellular and surface glycoproteins contain sialic acid (SA) as the monosaccharide located on the reducing end of the glycans. These can either be located on the glycoproteins as monomer, dimers, trimers or as large polysialic acid structures comprising of more than 200 SA residues. It has previously been demonstrated that cancer development and staging may be associated with a significant over-representation of SA on the cell surface glycoproteins of cancer cells compared to normal cells (e.g., [Dall'Olio, F., Chiricolo, M. Sialyltransferases in cancer. Glycoconj J. 2001, 18(11-12), 841-50]). The transformation of a tumor from benign to malignant is associated with increased SA content on the surface glycoproteins. Also it is well known that the amount of free SA and lipid and protein bound SA is elevated in plasma from cancer patients compared to healthy individuals.

Based on the methods developed in my group for selective isolation of sialylated peptides we have initiated a research program together with Professor Dr. Med. Henrik Ditzel, Medical Biotechnology Center, Odense University Hospital, Denmark and Dr. Med. Niels H.H. Heegaard, State Serum Institute, Denmark to investigate the role of glycosylation, especially sialylation, in cancer development. This is primarily a basic research project aiming at understanding the function of sialic acids and glycosylation in relation to cancer. In this project we will correlate the expression and activity of sialyltransferases in cancer cells and “normal” cells with the sialylation degree of the surface glycoproteins using stabile isotope labelling, TiO2 chromatography, hydrophilic interaction chromatography and mass spectrometry.

5: Biomarker discovery program

The aim of these clinical proteomics project is to investigate the possibility of detecting modified biomarkers (e.g., phosphorylated, glycosylated, oxidized etc.) indicative of a recent onset of T1DM and cancers in plasma. We have taken two different approaches for this research.

The primary focus of this research is to investigate if glycosylated proteins, especially sialylated glycoproteins can be used as biomarkers for a number of diseases including Type 2 diabetes, cancers, Alzheimers Disease and other CNS related diseases.